Thrombin is a multifunctional serine protease generated at sites of vascular injury, inflammation, or endothelial activation. In addition to its activity as a coagulation factor, it is a potent agonist for many cell types, including platelets, leukocytes, and endothelium. Its varied cellular activities suggest that in addition to its role in hemostasis, thrombin may play a role in inflammatory and proliferative responses to injury. A G protein- couple receptor that mediates thrombin activation of human platelets ha been cloned. Previous studies by our group had demonstrated that this receptor, protease-activated receptors-1 (PAR-1), is activated by a novel proteolytic mechanism. Another protease-activated receptor, PAR-2, subsequently was cloned by homology to PAR-1 and is activated by trypsin but not thrombin. Recently, the applicant disrupted the gene for PAR-1 in mice, which revealed PAR-1 plays an unexpected role in vasculogenesis in mouse embryonic development and that a second thrombin receptor exists in mouse platelets. We cloned the second thrombin receptor, termed PAR-3, which is homologous to PAR-1 and -2. The applicant is particularly interested in thrombin induced signaling in endothelial cells, because of their location at the blood/tissue interface and their diverse roles in vessel wall pathobiology. Preliminary studies for this project that demonstrated that all three PARs are expressed in endothelium, and the applicant has identified pharmacologically a third thrombin receptor that is expressed in a transformed endothelial cell line. The overall objective of this project is to elucidate the roles of the various protease-activated receptors in endothelial responses to thrombin in normal biology and disease. We propose to combine cellular and molecular biologic approaches in in vitro and in vivo studies as follows: Specific Aim 1: To clarify the molecular mechanisms of thrombin activation of endothelial cells in vitro: a) Expression: We will identify which thrombin receptors are expressed in various endothelial cells by Norther blot, Western blot, and immunofluorescence microscopy; b) Sufficient role: We will use specific agonists for PAR-1, -2, and -3 to identify which endothelial responses to thrombin can be mediated by activation of the PARs; c) Necessary roles: To identify which receptors are necessary for thrombin effects, we will use pharmacologic desensitization experiments, antibody blocking studies, and cultures of microvascular endothelial cells from mice with disruption of par-1, -2, or -3 genes. Specific Aim 2: To identify the roles of protease-activated receptors in endothelial responses to thrombin in vivo: a) Expression: We will identify which PARs are expressed in various endothelial cells in normal and diseased tissues by in situ hybridization and immunohistochemistry; b) Sufficient roles: We will use specific agonists for PARs to identify which vascular responses to thrombin can be mediated by activation of the PARs; c) Necessary roles: We will examines the vascular responses to thrombin and VEGF in par-1 -/- mice. We will further characterize the embryonic lethal phenotype in par-1 -/- mice, including an attempted rescue by transgenic expression of PAR-1 in endothelium. Specific Aim 3: To characterize and clone other protease-activated receptors that may mediate thrombin activation of endothelium: a) We will clone a third thrombin receptor from bEND.3 transformed mouse brain endothelial cells by expression cloning or homology-based approaches; b) We will characterize the expression pattern, protease sensitivity, and signaling properties of new thrombin receptors. Our hope is that a more complete understanding of thrombin stimulation of endothelium can be coupled with our increasing knowledge of the pathophysiology of the endothelium, in order to develop new strategies for therapeutic interventions in cardiovascular disease.